Synthesis, Radiolabeling, and In Vivo Imaging of PEGylated High

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Synthesis, Radiolabeling, and In Vivo Imaging of PEGylated HighGeneration Polyester Dendrimers Stuart A. McNelles, Spencer D. Knight, Nancy Janzen, John F. Valliant,* and Alex Adronov* Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario L8S 4M1, Canada

Downloaded by NEW YORK UNIV on September 9, 2015 | http://pubs.acs.org Publication Date (Web): August 31, 2015 | doi: 10.1021/acs.biomac.5b00911

S Supporting Information *

ABSTRACT: A fifth generation aliphatic polyester dendrimer was functionalized with vinyl groups at the periphery and a dipicolylamine Tc(I) chelate at the core. This structure was PEGylated with three different molecular weight mPEGs (mPEG160, mPEG350, and mPEG750) using thiol− ene click chemistry. The size of the resulting macromolecules was evaluated using dynamic light scattering, and it was found that the dendrimer functionalized with mPEG750 was molecularly dispersed in water, exhibiting a hydrodynamic diameter of 9.2 ± 2.1 nm. This PEGylated dendrimer was subsequently radiolabeled using [99mTc(CO)3(H2O)3]+ and purified to high (>99%) radiochemical purity. Imaging studies were initially performed on healthy rats to allow comparison to previous Tc-labeled dendrimers and then on xenograft murine tumor models, which collectively showed that the dendrimers circulated in the blood for an extended period of time (up to 24 h). Furthermore, the radiolabeled dendrimer accumulated in H520 xenograft tumors, which could be visualized by single-photon emission computed tomography (SPECT). The reported PEGylated aliphatic polyester dendrimers represent a new platform for developing tumor-targeted molecular imaging probes and therapeutics.

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be readily synthesized through convergent32,33 or divergent approaches,34 they exhibit low in vivo toxicity, are biocompatible, and are biodegradable.35−37 The latter two features are particularly important because macromolecules and nanoscale materials with very long circulation times must be biodegraded in order to prevent long-term accumulation. In addition, bisMPA dendrimers are commercially available. We have already shown that bis-MPA dendrimers of high generation can be prepared with an easily removable core protecting group that enables the introduction of a single dipicolyl amine (DPA) ligand at the dendrimer core.38 This allows for site-specific radiolabeling with 99mTc, the most commonly used γ-emitting radionuclide in diagnostic imaging.39 In our previous work, we produced a series of monodisperse, well characterized dendrimers up to the seventh generation, that contained a single 99mTc atom chelated at the core.38 Single photon emission computed tomography (SPECT) imaging allowed real-time in vivo monitoring of these bisMPA dendrimer structures upon injection into the bloodstream of rats. These studies showed that the circulation time of the dendrimers, regardless of generation, was extremely short, with nearly complete clearance from the blood into the bladder within 15 min postinjection.38 This rapid clearance was most likely the result of their low hydrodynamic diameter, as the highest generation structure had a diameter on the order of 3−

INTRODUCTION Macromolecular therapeutic agents have garnered increasing interest over the past four decades.1−7 It has been shown repeatedly that conjugation of pharmaceuticals to water-soluble, nontoxic, biocompatible polymers results in improved drug solubility, increased blood circulation time, and decreased systemic toxicity.8−10 In addition, the ability to introduce multiple functionalities on a macromolecule provides potential for site-specific targeting of disease.11−14 Of the available macromolecular architectures, dendritic macromolecules offer a number of unique advantages over their linear counterparts as scaffolds for conjugation of therapeutic agents.15−18 The precise, stepwise synthesis of dendrimers allows unparalleled structural precision relative to other synthetic macromolecules, enabling modification of the core, interior, or periphery in a highly controlled manner.19−21 The globular nature of highgeneration dendrimers additionally permits site isolation of moieties introduced at the core or interior, with macroscopic properties dictated by the surface functionalities that are introduced.22 This unique aspect of dendrimers opens the possibility for development of multifunctional dendritic structures capable of targeted simultaneous delivery of both therapeutic and diagnostic imaging agents, effectively resulting in a “theranostic” scaffold.23−26 Among the available dendrimer structures,27−29 aliphatic polyester dendrimers based on the 2,2-bis(hydroxymethyl)propanoic acid (bis-MPA) branching unit are ideal scaffolds for theranostic applications. 3 0 , 3 1 These poly(2,2-bis(hydroxymethyl)propanoic acid) (bis-MPA) dendrimers can © XXXX American Chemical Society

Received: July 8, 2015 Revised: August 15, 2015

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DOI: 10.1021/acs.biomac.5b00911 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

instrument and equilibrated to 37 °C for 5 min. Instrument parameters were set automatically during the beginning of the run, and samples were run in triplicate with the average hydrodynamic diameter and zeta potential calculated from the three runs. Zeta potential measurements were taken immediately after the DLS data using the same experimental conditions using a Malvern zeta potential dip cell. 99m TcO4− was eluted from a 99 Mo/99mTc generator using 4 mL of 0.9% saline in order to sufficiently concentrate the resulting 99mTcO4− for chelation to the dendrimers. Caution: 99mTc is radioactive and should only be handled in an appropriately licensed and equipped laboratory. Synthesis of Dipicolylamine (DPA) Ligand. Synthesis of tertButyl (4-Aminobutyl)carbamate (6). 1,4-Diaminobutane (0.5 g, 5.7 mmol) and t-butyl phenyl carbonate (1.0 g, 5.7 mmol) were added to a 50 mL flame-dried round-bottom flask and dissolved in 20 mL of anhydrous ethanol, which was refluxed under an argon atmosphere for 16 h. Solvent was removed in vacuo, then the residual oil was diluted with 20 mL of water and acidified to pH = 3 with 2 M HCl. This was extracted with CH2Cl2 (3 × 10 mL), then the aqueous phase was basified by the addition of 100 mL of 2 M NaOH. This was further extracted with CH2Cl2 (3 × 50 mL), dried over sodium sulfate, filtered on a glass frit, and dried in vacuo to leave a yellow oil (0.689 g, 65%). 1 H NMR (700 MHz; CDCl3): δ 1.43 (d, J = 11.1 Hz, 9H), 1.47−1.44 (m, 2H), 1.51 (dt, J = 14.6, 7.1 Hz, 2H), 2.70 (d, J = 13.7 Hz, 2H), 3.12 (d, J = 6.0 Hz, 2H), 4.63 (d, J = 145.1 Hz, 1H). 13C NMR (176 MHz; CDCl3): δ 27.63, 28.56, 31.02, 40.58, 41.97, 79.16, 156.13. MS Calcd for C9H20N2O2 [M]+ = 188.1525 and [M + H]+ = 189.1603. Found High Resolution ES MS [M + H]+ = 189.1603. Synthesis of tert-Butyl (4-(Bis(pyridin-2-ylmethyl)amino)butyl)carbamate (7). 2-Pyridinecarboxaldehyde (2.05 g, 19.2 mmol) and 6 (1.2 g, 6.4 mmol) were added to a 50 mL flame-dried round-bottom flask along with 20 mL of dry CH2Cl2. Acetic acid (0.036 mL, 0.64 mmol) was added, and the mixture was stirred for 4 h. The reaction mixture was cooled to 0 °C, and then sodium triacetoxyborohydride (4.73 g, 22.3 mmol) was added, producing a bright yellow color. After 3 h, the solution was diluted with 60 mL of CH2Cl2 and washed with 1 M NaOH (3 × 50 mL). The reaction mixture was dried over magnesium sulfate, filtered through a glass frit and dried down in vacuo. This was then purified by flash chromatography in 10% MeOH:DCM to give the product as a yellow oil (1.967 g, 83%). 1H NMR (700 MHz; CDCl3): δ 1.42 (s, 9H), 1.46 (q, J = 7.3 Hz, 2H), 1.56 (t, J = 7.4 Hz, 2H), 2.55 (s, 2H), 3.06−3.00 (m, 2H), 3.80 (s, 4H), 4.70 (d, J = 191.2 Hz, 1H), 7.13 (dd, J = 6.8, 5.5 Hz, 2H), 7.49 (d, J = 7.8 Hz, 2H), 7.64 (td, J = 7.6, 1.6 Hz, 2H), 8.52 (d, J = 4.7 Hz, 2H). 13C NMR (176 MHz; CDCl3): δ 24.41, 27.90, 28.59, 40.46, 54.07, 60.58, 122.04, 123.08, 136.49, 149.14, 156.12, 159.95. MS Calcd for C21H30N4O2 [M]+ = 370.2369, [M + H]+ = 371.2447. Found High Resolution ES MS [M + H]+ = 371.2441. Synthesis of N1,N1-Bis(pyridin-2-ylmethyl)butane-1,4-diamine (8). Monoboc-protected DPA ligand 7 (200 mg, 0.5 mmol) was dissolved in 6 mL of CH2Cl2. This was cooled to 0 °C, and 3 mL of trifluoroacetic acid was added dropwise and stirred for 3 h. This was basified by the dropwise addition of 2 M NaOH and extracted with CH2Cl2 (3 × 10 mL). The organic layer was then dried in vacuo, resulting in yellow/orange oil (114 mg, 79%). 1H NMR (600 MHz; CDCl3): δ 1.45−1.41 (m, 2H), 1.59−1.54 (m, 2H), 2.54 (s, 2H), 2.63 (t, J = 7.0 Hz, 2H), 3.80 (s, 4H), 7.14−7.13 (m, 2H), 7.51 (d, J = 7.8 Hz, 2H), 7.63 (dd, J = 7.6, 1.8 Hz, 2H), 8.52−8.51 (m, 2H). 13C NMR (151 MHz; CDCl3): δ 24.64, 31.49, 42.08, 54.39, 60.62, 122.07, 123.05, 136.53, 149.14, 160.03. MS Calcd for C16H22N4 [M]+ = 270.1844, [M + Na]+ = 293.1742. Found High Resolution ES MS [M + H]+ = 293.1734. Synthesis of mPEG750-SH. Synthesis of Allyl mPEG750 (9). PEG750 monomethyl ether (10 g, 13.3 mmol) and allyl bromide (2.42 g, 20 mmol) were added to a 300 mL round-bottom flask and dissolved in 100 mL of CH2Cl2. To this, 20 mL of saturated aqueous potassium hydroxide was added, and the reaction was left stirring. Immediately after addition of potassium hydroxide, the solution began to turn slightly yellow. After 1 h, the aqueous layer was removed in a separatory funnel and washed with CH2Cl2 (1 × 20 mL), which was added to the remaining organic phase. This was dried over sodium

4 nm. Considering that the renal clearance threshold is ca. 5 nm,40 it is not surprising that these structures were rapidly filtered from the bloodstream via the kidneys. This rapid clearance was problematic as it prevents dendrimer accumulation within tumors via the enhanced permeability and retention (EPR) effect; a well-documented passive tumor targeting method that depends on long blood circulation times.41−47 In order to lengthen dendrimer circulation time, it is necessary to increase the hydrodynamic diameter of the bisMPA dendrimers without affecting their water-solubility and biocompatibility. To achieve this, we embarked upon their surface functionalization with poly(ethylene glycol) chains, a strategy commonly referred to as PEGylation.48−54 PEGylation is known to increase the circulation time of proteins, macromolecules, and nanoparticles, allowing their passive uptake in tumors through the EPR effect. Two features of PEGylation are responsible for the increased circulation time in vivo, including an increase in the hydrodynamic diameter of the PEGylated species, which impedes clearance through the kidneys,55,56 and an increase in hydrophilicity of the structure, which reduces the ability of opsonins to bind to its surface.57−59 PEGylation is therefore considered an effective way to control the pharmacokinetics of various nanoscale structures in therapeutic applications, and has resulted in a number of PEGylated pharmaceuticals undergoing clinical trials and FDA approval.60,61 Here, we describe the preparation of highgeneration dendrimers that have been surface-PEGylated via the thiol−ene “click” reaction and subsequently radiolabeled with 99mTc at their core. These novel dendrimers have a hydrodynamic diameter above the renal clearance threshold and are shown to exhibit long circulation times when injected in healthy animal models. Upon injection into the bloodstream of nude mice bearing squamous cell (H520) xenograft tumors, these PEGylated bis-MPA dendrimers were found to accumulate in the tumor, allowing for visualization by SPECT imaging.

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MATERIALS AND METHODS

Materials. All chemicals used were sourced from Sigma-Aldrich and used as supplied without further purification, unless otherwise noted. 2-Pyridinecarboxaldehyde was freshly distilled prior to use. Structural Characterization. NMR spectroscopy was performed on either a Bruker AVANCE AV600 spectrometer at 600 MHz or a Bruker AVANCE AV700 at 700 MHz. CDCl3 and CD3OD were used as solvents, and the residual nondeuterated solvent signals were used as internal chemical shift references for both 1H and 13C spectra. MALDI spectra were acquired using a Bruker Ultraflextreme spectrometer in positive ion mode using dithranol as matrix. Exact masses were determined using a Micromass Q-TOF Global Ultima. Analytical HPLC was performed using a Waters 1525 Binary solvent pump using a Waters 2998 Photodiode Array detector and Bio-Rad IN/US γ -detector using a Phenomenex Luna C18(2) column (4.6 mm × 150 mm). The mobile phase consisted of chromatography grade water (A) and chromatography grade acetonitrile (B). The gradient protocol consisted of a 2 min hold at 100% solvent A, then a gradient to 50% B from 2 to 7 min, a 3 min hold at 50% B, and then a gradient to 100% B from 10 to 15 min, with a 3 min hold at 100% B, followed by a step gradient to 100% A, which was held for 5 min in order to equilibrate the column for subsequent runs. All HPLC runs were performed at a flow rate of 1 mL/min and were monitored at a wavelength of 254 nm. Dynamic light scattering data was collected on a Malvern Zetasizer Nano ZS instrument. A solution of 1 mg/mL of PEGylated dendrimer was made in 10 mM phosphate buffered saline, and this was filtered through a 0.22 μm Teflon syringe filter into disposable polystyrene cuvettes. This sample was then placed in the B


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Biomacromolecules sulfate, filtered through a glass frit and dried in vacuo, leaving the product as an off-white waxy solid (10.53 g, quant). 1H NMR (CDCl3, 600 MHz): δ 3.37 (s, 3H), 3.53−3.55 (m, 2H), 3.59−3.61 (m, 3H), 3.65 (q, J = 5.8 Hz, 61H), 4.02 (dt, J = 5.7, 1.4 Hz, 2H), 5.17 (dq, J = 10.4, 1.5 Hz, 1H), 5.25−5.28 (m, 1H), 5.91 (ddt, J = 17.2, 10.4, 5.7 Hz, 1H). Synthesis of mPEG750 Thioacetate (10). Allyl PEG750 monomethyl ether 9 (2.00 g, 2.53 mmol) was added to a 20 mL scintillation vial, along with 2,2-dimethoxy-2-phenylacetophenone (20 mg, 0.005 mmol). These were dissolved in 8 mL of HPLC-grade THF, and then thioacetic acid (250 mg, 3.2 mmol) was added. This was degassed with argon for 10 min, then irradiated with 365 nm UV light for 2 h. Solvent and excess thioacetic acid were removed in vacuo, leaving the product as a waxy yellow solid (2.12 g, 97%). 1H NMR (CDCl3, 600 MHz): δ 1.84 (quintet, J = 6.7 Hz, 2H), 2.31 (s, 3H), 2.93 (t, J = 7.2 Hz, 2H), 3.36 (s, 3H), 3.49 (t, J = 6.2 Hz, 3H), 3.53 (dd, J = 5.6, 3.8 Hz, 3H), 3.56 (dd, J = 5.9, 3.7 Hz, 3H) 3.62 (t, J = 9.1 Hz, 70H). Synthesis of mPEG750-SH (11). PEG750 monomethyl ether thioacetate 10 (2.12 g, 2.45 mmol) was added to a 50 mL roundbottom flask and put under an argon atmosphere. It was subsequently dissolved in 20 mL of deoxygenated 0.25 M NaOH, producing a yellow solution that rapidly turned orange, and stirred for 12 h. This was subsequently acidified with 1 M sodium bisulfate (10 mL), causing the solution to turn from orange to yellow. This was extracted with CH2Cl2 (3 × 15 mL), dried over sodium sulfate, filtered through a glass frit, and dried in vacuo to give the desired product as a waxy yellow solid (1.98 g, 99%). 1H NMR (CDCl3, 600 MHz): δ 1.37 (t, J = 8.0 Hz, 1H), 1.87 (quintet, J = 6.6 Hz, 2H), 2.61 (q, J = 7.5 Hz, 2H), 3.37 (s, 3H), 3.54 (dd, J = 5.7, 4.0 Hz, 3H), 3.58 (dd, J = 6.5, 4.2 Hz, 2H), 3.61−3.63 (m, 63H) 3.71−3.75 (m, 1H). Synthesis of Dendrimers. Synthesis of Pentenoic Acid Anhydride (2). Pentenoic acid (6.45 g, 64.6 mmol) and dicyclohexylcarbodiimide (6.82 g, 33.1 mmol) were added to a flame-dried 500 mL round-bottom flask along with 300 mL of CH2Cl2. This was left to stir overnight under an argon atmosphere, and the resulting mixture was filtered on a sintered glass frit to remove the solids. The filtrate was concentrated in vacuo to yield a viscous yellow oil (5.75 g, 98%). 1 H NMR (CDCl3, 600 MHz): δ 2.42 (dt, 4H, J = 7.2, 6.6), 2.56 (t, 4H, J = 7.5), 5.07 (m, 4H), 5.81 (m, 2H). Synthesis of pTSe-G5-(PTA)32 (3). pTSe-G5-(OH)32 (1.8 g, 0.5 mmol), pentenoic acid anhydride (5.5 g, 30.3 mmol), and 4dimethylaminopyridine (370 mg, 3.0 mmol) were added to a flamedried 50 mL round-bottom flask, along with 12 mL of CH2Cl2 and 4 mL of pyridine. This was stirred overnight, and the reaction mixture was washed with 1 M NaHSO4 (4 × 10 mL), 10% Na2CO3 (3 × 10 mL), and brine (1 × 10 mL). This was then dried over sodium sulfate, filtered through a sintered glass frit, and solvent was removed by rotary evaporation in vacuo. The resulting mixture was purified by silica gel chromatography using an eluent consisting of 10% ethyl acetate, 40% hexanes, and 50% CH2Cl2, which was increased to 20% ethyl acetate, 30% hexanes, and 50% CH2Cl2 after excess pentenoic anhydride had eluted. The fractions containing product were dried under reduced pressure and were collected as a viscous yellow oil (3.01 g, 97%). 1H NMR (700 MHz; MeOD): δ 1.36−1.26 (m, 93H), 2.34 (q, J = 7.0 Hz, 64H), 2.44 (t, J = 7.3 Hz, 64H), 2.48 (s, 3H), 3.62−3.61 (m, 2H), 4.35−4.20 (m, 124H), 4.52−4.51 (m, 2H), 4.98 (dd, J = 10.3, 1.3 Hz, 32H), 5.05 (dd, J = 17.2, 1.5 Hz, 32H), 5.86−5.80 (m, 32H), 7.49 (d, J = 8.2 Hz, 2H), 7.85 (d, J = 8.2 Hz, 2H). 13C NMR (176 MHz; MeOD): δ 18.18, 18.34, 18.41, 29.92, 34.29, 47.75, 48.06, 66.48, 66.63, 67.11, 67.25, 67.67, 116.19, 129.37, 131.37, 138.03, 173.09, 173.15, 173.56, 173.87. MS Calcd for C324H452O128S [M]+ = 6422.8581, [M + Na]+ = 6445.8479. Found MALDI-TOF MS [M + Na]+ = 6449.09. Synthesis of COOH-G5-(PTA)32 (4). pTSe-G5-(PTA)32 (1 g, 0.15 mmol) and 1,8-diazabicycloundec-7-ene (DBU) (0.25 mL, 1.5 mmol) were added to a flame-dried 100 mL round-bottom flask along with 50 mL of CH2Cl2 and stirred for 2 h. This was washed with 1 M NaHSO4 (2 × 20 mL), and solvent was removed by rotary evaporation in vacuo. Product was isolated via silica gel chromatography in 1:1 EtOAc/ hexanes, resulting in a clear colorless oil (900 mg, 93%). 1H NMR (700 MHz; MeOD): δ 1.38−1.28 (m, 100H), 2.36 (q, J = 7.0 Hz,

64H), 2.46 (t, J = 7.3 Hz, 64H), 4.38−4.23 (m, 124H), 5.01 (dd, J = 10.3, 1.5 Hz, 32H), 5.09−5.07 (m, 32H), 5.85 (ddt, J = 17.0, 10.4, 6.5 Hz, 32H). 13C NMR (176 MHz; MeOD): δ 18.33, 18.41, 29.92, 34.30, 47.92, 48.14, 66.48, 66.62, 67.08, 67.24, 67.92, 116.18, 138.03, 173.07, 173.16, 173.56, 173.88. MS Calcd for C315H442O126 [M]+ = 6240.8179, [M + H]+ = 6241.8251. Found MALDI-TOF MS [M + H]+ = 6246. Synthesis of NHS-G5-(PTA)32. Synthesis of COOH-G5-(PTA)32 (300 mg, 0.05 mmol), N-hydroxysuccinimide (22 mg, 0.2 mmol), and 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (30 mg, 0.2 mmol) were added to a 10 mL flame-dried round-bottom flask, to which 2 mL of dry CH2Cl2 was added. This was stirred overnight, and the resulting mixture was purified by column chromatography with 3:2 EtOAc/Hex. This was then dried in vacuo, resulting in a clear, colorless oil (302 mg, 99%). The product was used immediately after purification to ensure that the NHS ester did not hydrolyze. 1H NMR (600 MHz, CDCl3): δ 1.35−1.21 (m, 93H), 2.38−2.32 (m, 64H), 2.46−2.39 (m, 64H), 2.86 (s, 4H), 4.39−4.15 (m, 124H), 5.09−4.97 (m, 64H), 5.80 (tdd, J = 6.3, 10.4, 16.9 Hz, 32H). Synthesis of DPA-G5-(PTA)32 (5). NHS-G5-(PTA)32 (300 mg, 0.05 mmol), DPA ligand 8 (30 mg, 0.1 mmol), and triethylamine (0.07 mL, 0.25 mmol) were added to a 10 mL flame-dried round-bottom flask and dissolved in 5 mL of dry CH2Cl2. This was stirred at room temperature for 3 days, then purified via column chromatography on silica gel with 5% methanol in CH2Cl2. The product fractions were collected and dried in vacuo, resulting in a slightly yellow oil (298 mg, 97%). 1H NMR (MeOD, 700 MHz): δ 1.26−1.35 (m, 93H), 1.50− 1.55 (m, 2H), 1.59−1.64 (m, 2H), 2.34 (q, J = 6.8 Hz, 64H), 2.43 (t, J = 7.3 Hz, 64H), 2.58 (t, J = 7.0 Hz, 2H), 3.17−3.19 (m, 2H), 3.80 (s, 4H), 4.20−4.33 (m, 124H), 4.98 (d, J = 10.2 Hz, 32H), 5.05 (d, J = 17.2 Hz, 32H), 5.82 (dd, J = 17.0, 10.4 Hz, 32H), 7.28 (t, J = 6.2 Hz, 2H), 7.63 (d, J = 7.7 Hz, 2H), 7.79 (t, J = 7.6 Hz, 2H), 8.44 (d, J = 4.5 Hz, 2H). 13C NMR (176 MHz; MeOD): δ 18.42, 25.54, 28.50, 29.92, 34.30, 40.78, 47.74, 48.03, 48.05, 55.40, 61.05, 66.46, 66.61, 67.02, 67.17, 68.56, 116.19, 123.79, 124.81, 138.02, 138.61, 149.58, 173.07, 173.14, 173.55, 173.85. MS Calcd for C330H460N4O125 [M]+ = 6478.9761, [M+NH4]+ = 6497.0099. Found MALDI-TOF MS [M +NH4]+ = 6494.47. Synthesis of DPA-G5-PEG750 (12). DPA-G5-(PTA)32 (100 mg, 0.015 mmol) and mPEG750-SH 11 (1.21 g, 1.49 mmol) were added to a 20 mL scintillation vial, along with 2,2-dimethoxy-2-phenylacetophenone (7 mg, 0.025 mmol). To this, 8 mL of HPLC grade THF was added, and the reaction mixture was stirred until all components dissolved. This was sparged with argon with sonication for 15 min, then irradiated with UV at 365 nm for 24 h. After 24 h, an additional 7 mg aliquot of 2,2-dimethoxy-2-phenylacetophenone was added, and the reaction was irradiated for a further 72 h. Solvent was removed by rotary evaporation, and the resulting viscous orange oil was redissolved in ethanol. This was added dropwise to 500 mL of diethyl ether at 0 °C, then left to warm to room temperature. The product phase separated as a viscous oil on the bottom of the flask. The residual ether was decanted and the procedure was repeated to give the final product (201 mg, 40%). 1H NMR (CDCl3, 700 MHz): δ 1.28−1.34 (m, 105H), 1.63 (s, 56H), 1.72 (s, 50H), 1.83 (s, 53H), 2.39 (s, 67H), 2.58 (d, J = 32.6 Hz, 95H), 3.36 (s, 47H), 3.54−3.64 (m, 1H), 4.24−4.33 (m, 124H), 5.01−5.10 (m, 15H), 5.85 (s, 7H). MALDI-TOF MS produced a broad mass distribution centered around m/z = 18800 Da (PDI = 1.01) due to the polydispersity of PEG chains. Radiolabeling Procedures. Synthesis of 99mTc(CO)3(H2O)3. To a 5 mL Biotage microwave vial were added sodium potassium tartrate (22 mg, 0.078 mmol), sodium borate decahydrate (20 mg, 0.052 mmol), sodium carbonate (15 mg, 0.14 mmol), and potassium boranocarbonate (10 mg, 0.075 mmol), then the vial was sealed with a septum and purged with nitrogen gas for 15 min. Approximately 2960 MBq (80 mCi) of sodium pertechnetate (Na 99mTcO4) in 4 mL of 0.9% saline was added to the vial. This was heated in a microwave reactor to 110 °C for 4 min. This was cooled to room temperature, and then the pH was adjusted to 6.5 by the dropwise addition of 1 M HCl. C


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Biomacromolecules


Scheme 1. Synthesis of the Fifth Generation Dendrimer Bearing Vinyl Groups at the Periphery and the DPA Ligand at the Core

Synthesis of 99mTcDPA-G5-PEG750. To a 2 mL Biotage microwave vial was added 2 mg of BisPy-G5-PEG750 dissolved in 0.1 mL of water and 925 MBq (25 mCi) of [99mTc(CO)3(H2O)3]+ in 0.9% saline (1 mL) was added. The vial was sealed with a septum and purged for 10 min with nitrogen gas, then heated in a microwave reactor for 15 min at 120 °C. The reaction was cooled to room temperature, and 5 mg of histidine dissolved in 0.30 mL of 0.9% saline was added via syringe. The reaction mixture was heated to 150 °C in a microwave reactor for 5 min, and upon cooling, the entire reaction mixture was passed through three GE HiTrap desalting columns in series to isolate the high molecular weight products. The final mixture was dried on a centrifugal evaporator and redissolved in 0.5 mL of deionized water. The desired product was isolated by HPLC to yield the radiochemically pure labeled dendrimer. The collected HPLC eluate was dried using a centrifugal V10 evaporator (Biotage), and redissolved in sterile 0.9% saline to a concentration of 185 MBq/mL (5 mCi/mL). Biological and Imaging Procedures. Cell Lines. NCI-H520 (H520) cells derived from human lung squamous cell carcinoma were purchased from ATCC (HTB-182) and maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin.

Animal Studies. All animal studies were approved by the Animal Research Ethics Board at McMaster University. Mice, female CD1 nu/ nu ordered from Charles River Laboratories, were maintained under clean conditions in an established animal facility with 12 h light/dark cycles and given food and water ad libitum. Rats, male Copenhagen ordered from Charles River Laboratories, were maintained under specific pathogen-free (SPF) conditions in an established animal facility with 12 h light/dark cycles and given food and water ad libitum. Tumor Inoculation. Female 4−5 week old CD1 nu/nu mice were injected with 2.0 × 106 H520 cells in matrigel/DPBS (1:1) subcutaneously into the right flank. Imaging studies were conducted 2 weeks following tumor inoculation. Imaging Study. X-ray images were acquired using a conebeam XSPECT scanner (Gamma Medica, Northridge, U.S.A.) with a source voltage of 75 kVp and a current of 165 μA at the McMaster Centre for Preclinical and Translational Imaging. Projection data was acquired with 1024 projection angles (1184 × 1120 pixels, 0.100 mm pixels) and reconstructed using a Feldkamp cone beam backprojection algorithm in COBRA (Exxim Software, Pleasanton, CA, U.S.A.) into 512 × 512 × 512 arrays (0.155 mm isotropic voxels). A water-filled tube was included within each scan in order to convert the voxel values D


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to Hounsfield units (HU). SPECT images were acquired on an XSPECT system (Gamma Medica, Northridge, U.S.A.), using dual sodium iodide crystals in combination with low-energy, highresolution, parallel-hole collimators. A total of 64 projections over 360° were acquired with an energy window of 159 ± 10% keV and then reconstructed using an OS-EM iterative reconstruction method (2 iterations/8 subsets) into 82 × 82 × 82 arrays (1.463 mm isotropic voxels) using in-house software. CT images were compressed to a 2563 matrix (0.31 mm isotropic voxels). Fusion was achieved by a rigidbody (linear) transformation of the SPECT image, during which, it is interpolated and resampled to the same matrix dimensions and voxel size as the compressed CT image. Rats were injected with approximately 25 MBq in 0.9% NaCl via the tail vein. Dynamic images were collected from time of injection until 0.5 h postinjection, with images being acquired every 15 s. Whole body SPECT/CT images were acquired at 4 h (10 s projections) and 22 h (30 s projections) postinjection. Mice were injected with approximately 9 MBq in 0.9% NaCl via the tail vein. Whole body SPECT/CT images were acquired at 2 h (20 s projections) and 6 h (40 s projections) postinjection. Imaging analysis was completed using AMIDE software.

Figure 2. MALDI-TOF spectra of TSe-G5-PTA32 (top), COOH-G5PTA32 (middle), and DPA-G5-PTA32 (bottom).

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Scheme 2. Preparation of PEG750-SH

RESULTS AND DISCUSSION Synthesis of the fifth generation bis-MPA dendron (1) with hydroxyl groups at the periphery and a protected acid group at

Figure 3. MALDI-TOF mass spectrum of DPA-G5-(PEG750).

Figure 1. 1H NMR (700 MHz) spectra of TSe-G5-PTA32 (3, top), COOH-G5-PTA32 (4, middle), and DPA-G5-PTA32 (5, bottom), all in CD3OD. Insets show a magnified view of aromatic signals for 3 and 5.

the core was accomplished following our previously reported protocol.38 To PEGylate the periphery of this dendron, we chose to investigate the thiol−ene click reaction due to its orthogonal nature to the functional groups present in the dendron backbone, as well as its previous use in functionalizing high generation dendrimers.62−64 We therefore modified the periphery of dendron 1 with vinyl groups via reaction with

Figure 4. Number-average dynamic light scattering data for DPA-G5(PEG750).

pentenoic acid anhydride (PTA, 2) in a manner directly analogous to the dendrimer growth steps (Scheme 1). This E


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Scheme 3. Radiolabeling of DPA-G5-(PEG750)

Once the alkene-functionalized dendrimers were synthesized, thiol-terminated poly(ethylene glycol) (PEG-SH) was required for peripheral PEGylation. Three molecular weights of PEG-SH were synthesized, including triethylene glycol (PEG160), PEG350, and PEG750. This was accomplished by first alkylating the PEG-OH monomethyl ether with allyl bromide under phase transfer conditions (Scheme 2). The resulting MeOPEG-(alkene) was then treated with a slight excess of thioacetic acid using 2,2-dimethoxy-2-phenylacetophenone (DMPA) photoinitiator and irradiated with 354 nm light to produce thioacetate terminated PEG. Hydrolysis with 0.2 M KOH liberated the desired PEG-SH in 90% overall yield. Thiol−ene click reactions on DPA-G5-(PTA)32 were first attempted with the thiol derivative of triethylene glycol (TEGSH). It was found that with the short TEG-SH, the thiol−ene reaction resulted in complete functionalization of the dendrimer in 16 h, as confirmed by 1H NMR via disappearance of the alkene protons. Complete functionalization with the longer PEG350-SH required a higher concentration of PEG350SH (0.25 M vs 0.1 M with TEG-SH) and a longer reaction time of 48 h, again confirmed by 1H NMR. However, when performing the thiol−ene reaction with PEG750-SH, complete functionalization could not be achieved. Based on the integration of the residual alkene protons in the 1H NMR, it was determined that a maximum of 24 of the 32 peripheral alkene groups were converted. Attempts to drive the reaction to higher conversion by increasing the amount of PEG750-SH, photoinitiatior, or reaction time, were unsuccessful. We attribute the inability to fully functionalize the periphery with PEG750-SH to the extreme steric bulk around the residual alkene sites once most of the periphery is covered with PEG750SH. Interestingly, MALDI-TOF MS analysis of the product (Figure 3) indicated an Mn of 18800 (PDI = 1.01), which corresponds to only 16 PEG750-SH chains at the dendrimer periphery. This underestimation likely results from a bias toward lower molecular weight structures, which are easier to ionize and ablate into the gas phase during the MALDI-TOF measurement. Dynamic light scattering (DLS) was used to characterize the hydrodynamic diameter of each of the PEGylated dendrimers. For both the DPA-G5-(TEG) and DPA-G5-(PEG350), DLS

Figure 5. HPLC chromatograms of DPA-G5-(PEG750) (A, UV detection) and [99mTcDPA-G5-(PEG750)]+ (B, gamma detection).

procedure resulted in quantitative conversion of the 32 peripheral hydroxyl groups in 95% isolated yield. After functionalizing the periphery with alkenes, the paratoluenesulfonylethanol (pTSe) core was removed by treating the dendron with an excess of DBU in dichloromethane, which allowed quantitative conversion to the carboxylic acid (Scheme 1). The resultant carboxylic acid was then activated by forming the N-hydroxysuccinimidyl ester, followed by efficient amidation with an aminobutyldipicolylamine ligand used for chelating [99mTc(CO)3]+, which was synthesized according to a previously reported procedure.38 Each of these structures was characterized by NMR (Figure 1), as well as MALDI-TOF mass spectrometry (Figure 2), which shows the conversion from the protected TSe-G5-(PTA)32 (3) to the deprotected acid at the core (4) and, finally, to the ligand-functionalized structure (5). F


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Figure 6. 2-D Scintigraphic images from the dynamic scanning study of [99mTcDPA-G5-PEG750]+ in rats. Each of the images shown covers 30 s beginning in the top left during the course of the first 15 min after injection.

Figure 7. Scintigraphic-CT images of [99mTcDPA-G5-(PEG750)]+ at 2 h postinjection (left) and 21.5 h postinjection (right).

showed that the dendrimers were not completely soluble in water, as they were found to aggregate to particle sizes of ∼200 nm (data not shown). However, the DPA-G5-(PEG750) was found to be readily soluble, giving a hydrodynamic diameter of 9.2 ± 2.1 nm (Figure 4). This is significantly larger than the renal clearance threshold, so we expected this structure to exhibit a long circulation time in vivo, despite incomplete functionalization of its periphery. Additionally, the zeta potential was measured and found to be +3.11 mV, which is very close to electrically neutral and is thus not expected to strongly interact with charged groups in various tissues and organs (specifically, the liver and kidneys). Radiolabeling of DPA-G5-(PEG750) with 99mTc was accomplished using the technetium tricarbonyl tris aqua [99mTc(CO)3(H2O)3]+ species, generated by the reduction of sodium pertechnetate (Na99mTcO4) using a modified procedure based on the work of Alberto and co-workers.65−67 Addition of [99mTc(CO)3(H2O)3]+ to an aqueous solution of DPA-G5-

Figure 8. Scintigraphic-CT image of H520 tumor model 6 h postinjection of [99mTcDPA-G5-(PEG750)]+ (9.2 MBq): H, heart; L, lungs; B, bladder; T, tumor.

(PEG750) in a microwave vial was followed by purging the headspace of the vial with nitrogen for 10 min and microwave heating to 120 °C for 15 min to yield [99mTcDPA-G5(PEG750)]+ (Scheme 3). Since both the polyester backbone of the dendrimer and the PEG chains can weakly coordinate [99m Tc(CO)3]+, the reaction mixture was subsequently challenged with 5 mg of histidine in deionized water and heated using microwave irradiation to 150 °C for 5 min. The resulting mixture was passed through three SEC HiTrap desalting cartridges (GE Healthcare), which completely removed 99mTc not bound to the chelate. This was verified by a second histidine challenge on the purified material, which G


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long-circulating dendrimers are able to accumulate in tumors, most likely via the EPR effect. It should be noted that, upon excision of the tumors after imaging, we found that the one mouse that did not show tumor uptake during SPECT imaging had a tumor that was approximately half the volume of the other two. This small tumor was likely poorly vascularized in comparison to the larger tumors, and so accumulation of the dendrimer within that tumor was impaired. While additional validation studies are needed in a larger cohort of animals, this work opens the possibility for using the PEGylated dendrimer reported here as a theranostic scaffold for delivering chemotherapeutic agents to tumors via the EPR effect, while simultaneously imaging the delivery process by SPECT-CT.

showed no radiolabeled histidine by HPLC. The proportion of loosely bound 99mTc was approximately 40%, based on the activity remaining in the collected fractions after size exclusion chromatography. HPLC characterization of the purified, radiolabeled dendrimer, [99mTcDPA-G5-(PEG750)]+, indicated that there was no measurable change in the retention time of the radiolabeled dendrimer compared to the free ligand, which is expected due to the peripheral mPEG750 chains that dominate the polarity and properties of these structures (Figure 5). The slight narrowing in the peak, as well as its slightly shifted retention time, is likely due to the significantly greater amount of material required to detect the unlabeled dendrimer with the UV detector compared to that needed to visualize the product in the gamma trace. After purification, [TcDPA-G5-PEG750]+ was produced in 14% ± 2% radiochemical yield in >99% radiochemical purity. Although the final yield of product was low, the synthesis can be routinely carried out starting with 925 MBq (25 mCi) of Na99mTcO4 eluted from the generator, which produces sufficient quantities of labeled compound for SPECT imaging studies. Imaging Studies. SPECT imaging was performed using a Gamma Medica X-SPECT system, where the labeled dendrimer (25 MBq, 700 μCi) in 0.9% saline was injected into the tail vein of two anesthetized male Copenhagen rats. Copenhagen rats were chosen in order to allow for a direct comparison of circulation time with our previous studies using bis-MPA dendrimers with hydroxyl groups at their periphery.38 The biodistribution was monitored by a dual head detector system with high-resolution parallel beam collimators, having a reconstruction field of view of 125 × 125 × 125 mm, with 3−4 mm spatial resolution. Individual images were collected every 15 s for the first 30 min of the dynamic scan. At 30 min, 2 h, and 22 h, SPECT scans were acquired with 10 s projections at the 2 h time point and 30 s projections at the 22 h time point. Figure 6 shows individual images from the dynamic scan, each produced from 30 s acquisitions (see Supporting Information for the corresponding time-lapse video). From these images, it is clearly visible that the majority of the injected activity remains in the blood pool, the heart, and the lungs, with a small amount of activity being excreted into the kidneys. At the 2 h time point, activity is predominantly seen in the heart and lungs again, with very little activity remaining in the bladder (Figure 7). At 22 h, activity is seen throughout the abdomen, and very little remains in the heart and lungs (Figure 7). This seems to indicate clearance of the dendrimer from the blood to the lymphatic system as well as the liver and spleen, which have previously been identified as a method of nanoparticle elimination.12,29 Having confirmed that the circulation time of the PEGylated dendrimers increased dramatically over that of unPEGylated dendrimers and was sufficiently long to allow tumor uptake via the EPR effect, preliminary tumor imaging studies were undertaken. Three CD1 nude mice, bearing two week old subcutaneous squamous cell (H520) tumors on their right flank, were injected, via the tail vein, with approximately 7.5− 10.4 MBq (200−280 μCi) of [99mTcDPA-G5-(PEG750)]+, and SPECT images were acquired at 2 and 6 h postinjection. After 2 h, most of the injected dose remained in circulation, with little uptake of the [TcDPA-G5-(PEG750)]+ observed in the tumors. However, at the 6 h time point, uptake in the tumors was clearly visible in two of the three mice (Figures 8). This provides compelling evidence that the radiolabeled, PEGylated,

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CONCLUSION In this work, high generation bis-MPA dendrimers were synthesized with a dipicolyl amine chelate at the core and alkene functionalities at the periphery. These alkene-terminated dendrimers were PEGylated via thiol−ene “click” coupling using PEG750-SH, resulting in PEGylated high-generation dendrimers with a near-zero zeta potential and a hydrodynamic diameter of ∼9 nm, slightly above the renal clearance threshold. These structures could be radiolabeled with 99mTc(CO)3 to produce dendritic SPECT imaging agents. Injection of these agents into healthy Copenhagen rats and imaging via SPECT indicated that they circulated in the blood for up to 24 h, making them acceptable for tumor delivery via EPR. When imaging mouse models inoculated with H520 tumor cells, dendrimer accumulation within the tumor was clearly evident 6 h postinjection. This work demonstrates that PEGylation of the fifth-generation bis-MPA dendrimer scaffold with PEG750 chains produces structures that are large and hydrophilic enough to exhibit extended blood circulation times and avoids rapid renal clearance, opening the possibility for accumulation within tumors through the EPR effect.

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ASSOCIATED CONTENT

* Supporting Information S

Time-lapse videos of dendrimer circulation within healthy Copenhagen rats are provided. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00911. (MPG) (MPG)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Natural Science and Engineering Research Council of Canada (NSERC), the NSERC CREATE Training Program on Molecular Imaging Probes (cMIP), and the Ontario Institute for Cancer Research (OICR).

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REFERENCES

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Synthesis, Radiolabeling, and In Vivo Imaging of PEGylated High

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